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Nuovi meccanismi regolativi ed attività del fattore trascrizionale NF-Y: prospettive per la terapia anti-tumorale.
No full textIl fattore di trascrizione NF-Y è composto da tre subunità altamente conservate, NF-YA, NF-YB e NF-YC, tutte necessarie per il legame sequenza-specifico al DNA. NF-Y ha un ruolo fondamentale nella regolazione della progressione del ciclo cellulare, controllando in particolare la trascrizione dei geni G2/M, ma anche di geni associati alla replicazione e al riparo del DNA.
Numerosi risultati evidenziano che NF-Y è coinvolto nella trasformazione tumorale. Sebbene nei tumori non siano mai state identificate specifiche mutazioni nelle subunità di NF-Y, l’esame sistematico dei profili d’espressione proteica indica che NF-YA aumenta in diversi tipi di cancro. Inoltre, l’analisi bioinformatica dei meccanismi regolativi perturbati nei tumori ha individuato in NF-Y uno dei fattori trascrizionali responsabili delle modificazioni che portano a trasformazione neoplastica.
L’aumento dei livelli d’espressione del gene regolato da NF-Y Topoisomerasi-IIα (TOP2A) è stato riscontrato in diverse cellule tumorali. L’enzima TOP2A svolge un ruolo fondamentale nel metabolismo del DNA e nell’organizzazione della cromatina. Questo enzima è il bersaglio di molti farmaci anti-tumorali di successo, come i cosiddetti “veleni della TOP2A”, che intrappolano i complessi TOP2A-DNA e creano rotture del doppio filamento di DNA (DSB), portando a morte cellulare. In questo lavoro mostriamo che il curcuminoide bis-DemetossiCurcumina (bDMC) induce DSB irreversibili e morte cellulare apoptotica selettivamente nelle cellule tumorali, bersagliando l'attività e l’espressione della TOP2A. Una breve esposizione a bDMC blocca i complessi TOP2A-DNA, inibendo l'attività decatenante dell’enzima, mentre l'esposizione prolungata reprime la trascrizione della TOP2A, riducendo il legame di NF-Y sul suo promotore ed alterando così la sua conformazione cromatinica. Infatti, bDMC compromette chiaramente l’espressione e la localizzazione sub-cellulare delle subunità di NF-Y. I nostri risultati suggeriscono che la duplice attività di bDMC su TOP2A rappresenta una nuova strategia terapeutica per indurre apoptosi persistente nelle cellule tumorali ed individuano la modulazione di NF-Y come un approccio promettente nella terapia anti-cancro.
Con l'obiettivo di poter sviluppare una terapia diretta contro NF-Y, abbiamo studiato i meccanismi molecolari che controllano l'espressione delle subunità di NF-Y in cellule tumorali. I nostri studi evidenziano un complesso processo di autoregolazione che controlla la corretta espressione NF-Y, sia a livello di trascrizione genica che di stabilizzazione proteica mediante interazioni proteina-proteina.
Infine, considerando che i livelli nucleari di NF-YA aumentano all'inizio della fase di Sintesi e che la sua inattivazione altera la progressione delle cellule attraverso la fase S, abbiamo studiato il ruolo di NF-Y nella replicazione del DNA. Trascrizione e replicazione del DNA sono processi fondamentali che devono essere regolati accuratamente per mantenere l'integrità del genoma ed è sempre più evidente che i fattori trascrizionali possono essere coinvolti nella regolazione della replicazione del DNA. Grazie al sistema di Xenopus laevis e a metodiche in vitro, abbiamo potuto studiare la replicazione del DNA in assenza di trascrizione, scoprendo un ruolo non-trascrizionale di NF-Y nella replicazione del DNA. In cellule umane, il silenziamento genico di NF-YA conferma che NF-Y è coinvolto nella replicazione del DNA e ulteriori studi potranno caratterizzare meglio quali sono i meccanismi molecolari alla base di questa attività.
Complessivamente, i nostri risultati evidenziano che NF-Y ha un ruolo a livello sia trascrizionale che non trascrizionale nel controllo della proliferazione cellulare, facendo di NF-Y un bersaglio ancora più interessante per lo sviluppo di nuove terapie anti-proliferative contro le cellule tumorali.The transcription factor NF-Y is a heterotrimeric complex composed by three highly conserved subunits, NF-YA, NF-YB and NF-YC, all necessary for sequence-specific DNA binding. NF-Y is a fundamental regulator of cell proliferation and cell cycle progression, controlling particularly the transcription of G2/M genes, but also genes associated to DNA replication and repair.
Numerous findings highlight that NF-Y is involved in cancer. Although mutations in NF-Y subunits have never been specifically identified in tumours, systematic examination of protein expression profiles indicates that NF-YA is upregulated in different types of cancer. In addition, the analysis of global regulatory perturbations across human cancers pointed at NF-Y as one of the transcription factors responsible for oncogenic transcriptional changes.
Increased levels of the NF-Y-target gene Topoisomerase-IIα (TOP2A) have been identified in various cancer cells. TOP2A enzyme plays a fundamental role in DNA metabolism and chromatin organization. It is the target of many successful anti-cancer drugs, such as TOP2A poisons which stabilize TOP2A-DNA cleavage complexes and create DNA double strand breaks (DSBs), leading to cell death. Here we show that the Curcumin-derivative bis-DemethoxyCurcumin (bDMC) irreversibly induces DSBs and apoptotic cell death selectively in cancer cells by targeting TOP2A activity and expression. Short-term exposure to bDMC induces retention of TOP2A-DNA intermediates, impairing TOP2A decatenating activity, while longer exposure inhibits TOP2A transcription by reducing the recruitment of NF-Y on its promoter, thus altering its chromatin status. Indeed, bDMC clearly impairs expression and sub-cellular localization of NF-Y subunits. Our findings suggest that the dual activity of bDMC on TOP2A represents a novel therapeutic strategy to induce persistent apoptosis in cancer cells and identify NF-Y regulation as a promising approach in anti-cancer therapy.
With the aim of developing NF-Y-targeted therapy, we investigated the molecular mechanisms controlling NF-Y subunits expression in cancer. Our studies highlight a complex autoregulatory loop which controls proper NF-Y expression, both at the level of gene transcription and of protein stabilization via protein-protein interactions.
Finally, taking into account that NF-YA nuclear levels increase at the onset of S phase and its inactivation affects the progression of cells through S phase, we investigated the role of NF-Y in DNA replication. Transcription and DNA replication are fundamental DNA metabolic processes that need to be finely regulated to preserve genome integrity and accumulating evidences suggest the involvement of transcription factors in the regulation of DNA replication. We exploited the Xenopus laevis cell-free system to study cell-cycle regulated DNA replication in the absence of transcription, unravelling a novel non-transcriptional role of NF-Y in DNA replication. In human cells, silencing of NF-YA confirms that NF-Y is involved in DNA replication and further studies will better characterize which are the underlying molecular mechanisms.
All together, our results highlight that NF-Y plays both a transcriptional and non-transcriptional role in controlling cell proliferation, making of NF-Y an even more attractive target for the development of anti-proliferative therapy in cancer cells
The interplay between NF-Y, AR and lipid metabolism regulates tumor aggressiveness in prostate cancer.
No full textProstate cancer (PCa) is the second most frequent cancer in Western men. Computational analyses linked the transcription factor NF-Y to progression from benign to localized PCa, aggressive signatures, response to androgen deprivation therapy (ADT) and metastasis.
The NF-YA gene encodes two alternatively spliced transcripts, NF-YAs and NF-YAl. We demonstrated that PCa samples are characterized by increased NF-YA levels, as well as higher NF-YAs/NF-YAl transcriptional ratio. In vitro and in vivo, NF-YA depletion affects PCa tumorigenicity and changes in NF-YA isoforms expression are associated with key clinical and molecular features of aggressive PCa. NF-YAs enhances tumor growth and metastasis, while NF-YAl increases cell motility. Stratification of patients based on NF-YAs expression is predictive of clinical outcome, although a significant decrease in the NF-YAs/NF-YAl ratio characterizes PCa circulating cells.
PCa cells depend on male sex hormones for growth and survival, which is the basis of ADT. While ADT is initially effective, patients eventually relapse with castration-resistant prostate cancer (CRPC). One of the cellular processes most affected by androgens is lipid metabolism, whose rewiring contributes to PCa development, progression, metastasis and recurrence. Androgens stimulate de novo lipogenesis and lipid uptake by activating SREBPs, the master transcription factors of cholesterol and fatty acid biosynthesis. SREBPs interact with target promoters in cooperation with NF-Y, which controls de novo lipid biosynthesis pathway. A feedforward mechanism between SREBP and the Androgen Receptor (AR) was also described.
We modulated NF-YA in androgen-sensitive healthy and cancer cell lines, performing cellular and molecular studies. RNA-seq analysis highlighted the alteration of lipid and cholesterol metabolic pathways and of the unfolded protein response (UPR) in NF-YAl-overexpressing cells. Bioenergetics metabolic profiling of NF-YA transduced cells confirmed the key role of NF-YAl in PCa cell metabolism. We found a reciprocal regulation of AR and NF-Y that relies in particular on NF-YAl.
Our data suggest that NF-Y, and specifically the longer NF-YA isoform, can participate in ADT responsiveness and resistance mediated by metabolic alterations. The characterization of these pathways could be useful in the stratification of PCa patients into sub-categories with different levels of aggressiveness and ADT sensitivity
Histone Marks-Dependent Effect on Alternative Splicing: New Perspectives for Targeted Splicing Modulation in Cancer?
Full text linkAlternative splicing (AS) is a tightly regulated mechanism that generates the complex human proteome from a small number of genes. Cis-regulatory RNA motifs in exons and introns control AS, recruiting positive and negative trans-acting splicing regulators. At a higher level, chromatin affects splicing events. Growing evidence indicates that the popular histone code hypothesis can be extended to RNA-level processes, such as AS. In addition to nucleosome positioning, which can generate transcriptional barriers to shape the final splicing outcome, histone post-translational modifications can contribute to the detailed regulation of single exon inclusion/exclusion. A histone-based system can identify alternatively spliced chromatin stretches, affecting RNAPII elongation locally or recruiting splicing components via adaptor complexes. In tumor cells, several mechanisms trigger misregulated AS events and produce cancer-associated transcripts. On a genome-wide level, aberrant AS can be the consequence of dysfunctional epigenetic splicing code, including altered enrichment in histone post-translational modifications. This review describes the main findings related to the effect of histone modifications and variants on splicing outcome and how a dysfunctional epigenetic splicing code triggers aberrant AS in cancer. In addition, it highlights recent advances in programmable DNA-targeting technologies and their possible application for AS targeted epigenetic modulation
The transcription factor NF-Y is required for satellite stem cell proliferation and skeletal muscle tissue repair
No full textThe transcription factor NF-Y, composed by NF-YA, NF-YB and NF-YC subunits, has an important role in the regulation of cellular proliferation and differentiation in different cell types, among which muscle cells. While NF-YA, the DNA binding subunit of NF-Y, is down-regulated in the adult muscle of WT mice, its expression is observed in the mdx mouse, model for Duchenne Muscular Dystrophy, in which a massive regeneration mediated by resident muscle stem cells, namely Satellite Cells (SCs), occurs. With the aim to investigate the role of NF-YA in the SCs proliferation and differentiation, we generated and characterized a conditional knock out mouse model in which NF-YA is deleted in Pax7+ SCs by Tamoxifen induction in adult NF-YAflox/flox:Pax7CreER mice (NF-YA cKO). Cellular and molecular analysis carried out on isolated myofibers and SCs from WT and NF-YA cKO mice highlighted that NF-Y activity is important for the maintenance of SCs homeostasis. NF-YA loss depletes Pax7+ SCs pool and impairs SCs proliferation. Moreover, SCs-mediated regeneration following muscle damage induced by cardiotoxin is delayed in NF-YA cKO. The effect of NF-YA abrogation was also explored in post-natal muscle growth. Immunohistological analysis showed defects in muscle morphology and a decrease in SCs number in 3 weeks aged NF-YA cKO mice, period of major increment of muscle mass by SCs-mediated myonuclear accretion. The molecular mechanism underlying the impairment of SCs activity following NF-YA loss was investigated by AdenoCre-induced NF-YA deletion in ex vivo cultured SCs. Overall, our results highlight a role of NF-Y in muscle regeneration and in SCs fate, whose modulation could be useful to improve stem cell based therapies to treat muscular dystrophies
Nuclear Estrogen Receptors in Prostate Cancer: From Genes to Function
Full text link: Estrogens are almost ubiquitous steroid hormones that are essential for development, metabolism, and reproduction. They exert both genomic and non-genomic action through two nuclear receptors (ERα and ERβ), which are transcription factors with disregulated functions and/or expression in pathological processes. In the 1990s, the discovery of an additional membrane estrogen G-protein-coupled receptor augmented the complexity of this picture. Increasing evidence elucidating the specific molecular mechanisms of action and opposing effects of ERα and Erβ was reported in the context of prostate cancer treatment, where these issues are increasingly investigated. Although new approaches improved the efficacy of clinical therapies thanks to the development of new molecules targeting specifically estrogen receptors and used in combination with immunotherapy, more efforts are needed to overcome the main drawbacks, and resistance events will be a challenge in the coming years. This review summarizes the state-of-the-art on ERα and ERβ mechanisms of action in prostate cancer and promising future therapies
Transcription Factors in Cancer: When Alternative Splicing Determines Opposite Cell Fates
Full text linkAlternative splicing (AS) is a finely regulated mechanism for transcriptome and proteome diversification in eukaryotic cells. Correct balance between AS isoforms takes part in molecular mechanisms that properly define spatiotemporal and tissue specific transcriptional programs in physiological conditions. However, several diseases are associated to or even caused by AS alterations. In particular, multiple AS changes occur in cancer cells and sustain the oncogenic transcriptional program. Transcription factors (TFs) represent a key class of proteins that control gene expression by direct binding to DNA regulatory elements. AS events can generate cancer-associated TF isoforms with altered activity, leading to sustained proliferative signaling, differentiation block and apoptosis resistance, all well-known hallmarks of cancer. In this review, we focus on how AS can produce TFs isoforms with opposite transcriptional activities or antagonistic functions that severely impact on cancer biology. This summary points the attention to the relevance of the analysis of TFs splice variants in cancer, which can allow patients stratification despite the presence of interindividual genetic heterogeneity. Recurrent TFs variants that give advantage to specific cancer types not only open the opportunity to use AS transcripts as clinical biomarkers but also guide the development of new anti-cancer strategies in personalized medicine
A non transcriptional role for NF-Y in DNA replication
No full textThe heterotrimeric transcription factor NF-Y, composed by NF-YA, NF-YB and NF-YC subunits, is a key transcriptional regulator of cell cycle progression.
Using data generated by ENCODE, we identified a striking overlap between loci bound by NF-Y-and ORC2, hinting at a possible role of NF-Y in DNA replication.
NF-YA knock-down leads to replication defects and the activation an intra-S checkpoint.
We investigated the role of NF-Y in DNA replication by using the Xenopus cell-free system. NF-Y subunits were found to be recruited to chromatin during DNA replication. Both immunodepletion of NF-YA or NF-YB and overexpression of a dominant-negative NF-YA mutant lead to a clear decrease in DNA synthesis.
In mammalian cells, NF-Y colocalizes and directly interacts with DNA replication proteins. Nascent strand abundance assay in NF-YA inactivated cells corroborates that NF-Y participates to the DNA replication process.
Our data highlight that, in addition to its transcriptional activity in controlling cell proliferation, NF-Y plays a key role in the non transcriptional control of DNA replication
Concurrent inhibition of enzymatic activity and NF-Y-mediated transcription of Topoisomerase-IIα by bis-DemethoxyCurcumin in cancer cells
No full textTopoisomerase-IIa (TOP2A) enzyme is essential for cell viability due to its fundamental role in DNA metabolism and in
chromatin organization during interphase and mitosis. TOP2A expression is finely regulated at the transcriptional level through
the binding of the CCAAT-transcription factor NF-Y to its promoter. Overexpression and/or amplification of TOP2A have been
observed in many types of cancers. For this reason, TOP2A is the target of the most widely successful drugs in cancer
chemotherapy, such as TOP2A poisons, which stabilize TOP2A-DNA cleavage complexes and create DSBs, leading to
chromosome damage and cell death. We previously reported that the Curcumin-derivative bis-DemethoxyCurcumin (bDMC) is an anti-proliferative agent that inhibits cell growth by concomitant G1/S and G2/M arrest. Here we showed that bDMC irreversibly induces DSBs in cancer cells, but not in normal cells, by targeting TOP2A activity and expression. TOP2A ablation by siRNA corroborates its contribution to apoptosis induced by bDMC. Short-term exposure to bDMC induces retention of TOP2A-DNA intermediates, while longer exposure inhibits TOP2A transcription by affecting expression and sub-cellular localization of NF-Y subunits. ChIP analysis highlighted reduced recruitment of NF-Y to TOP2A regulatory regions, concomitantly to histone
deacetylation and decreased gene transcription. Our findings suggest that the dual activity of bDMC on TOP2A represents a
novel therapeutic strategy to induce persistent apoptosis in cancer cells and identify NF-Y regulation as a promising approach in anti-cancer therapy
Understanding the molecular mechanisms of Curcumin derivatives chemotherapic activity
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A specific role for the splice variants of the transcription factor NF-Y in modulating the transcriptional activity of the myogenic program
No full textCell proliferation and differentiation programs are highly regulated transcriptional processes essential for myogenesis. The transcription factor NF-Y has been long considered a fundamental player of cell growth by supporting the basal transcription of various cell cycle genes. It is composed by the NF-YB/NF-YC heterodimer and NF-YA, which interacts with the other two subunits and confers the strict sequence specificity to the complex. The NF-YA gene encodes two alternatively splice transcripts (NF-YAs and NF-YAl), which differently regulate cell proliferation and differentiation, as shown in haematopoietic and mouse embryonic stem cells.
NF-YAl expression is down-regulated in terminally differentiated muscle cells and in skeletal and cardiac muscle tissues. Its forced expression in muscle cells committed to differentiate impairs their exit from the cell cycle and indirectly interferes with the differentiation program. Here we show that the two NF-YA isoforms play a different role in the transcriptional activity of the myogenic program and may regulate the activity of muscle satellite cells
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